Formation of l-alanine as a reduced end product in carbohydrate fermentation by the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus was found to form substantial amounts of l-alanine during batch growth on either cellobiose, maltose or pyruvate. Acetate, CO₂ and H₂ were produced next to alanine. The carbon- and electron balances were complete for all three substrates. Under standard growth conditions (N₂/CO₂ atmosphere) an alanine/acetate ratio of about 0.3 was found for either substrate. The alanine /acetate ratio was influenced, however, by the hydrogen partial pressure. In the presence of S⁰ or in coculture with Methanococcus jannaschii this ratio was only 0.07, whereas under a H₂/CO₂ atmosphere this ratio could amount up to 0.8. Alanine formation was also aflected by the NH_inf4^sup+concentration, i.e. below 4 mM, NH_inf4^sup+becomes limiting to alanine formation. Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation). The alanine aminotransferase probably cooperates with glutamate dehydrogenase (up to 23 U/mg; 90°C) and ferredoxin: NADP⁺ oxidoreductase (up to 0.7 U/mg, using methyl viologen; 90°C) to recycle the electron acceptors involved in catabolism. Thus, the existence of this unusual alanine-forming branch enables P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.Abbreviations DTT dithiothreitol - MV methyl viologen - AAT alanine aminotransferase - GDH glutamate dehydrogenase - MV: NADP⁺ OR methyl viologen: NADP⁺ oxidoreductase